Abstract

Analyzing microRNAs (miRNAs) within urine extracellular vesicles (EVs) is important for realizing miRNA-based, simple, and noninvasive early disease diagnoses and timely medical checkups. However, the inherent difficulty in collecting dilute concentrations of EVs (<0.01 volume %) from urine has hindered the development of these diagnoses and medical checkups. We propose a device composed of nanowires anchored into a microfluidic substrate. This device enables EV collections at high efficiency and in situ extractions of various miRNAs of different sequences (around 1000 types) that significantly exceed the number of species being extracted by the conventional ultracentrifugation method. The mechanical stability of nanowires anchored into substrates during buffer flow and the electrostatic collection of EVs onto the nanowires are the two key mechanisms that ensure the success of the proposed device. In addition, we use our methodology to identify urinary miRNAs that could potentially serve as biomarkers for cancer not only for urologic malignancies (bladder and prostate) but also for nonurologic ones (lung, pancreas, and liver). The present device concept will provide a foundation for work toward the long-term goal of urine-based early diagnoses and medical checkups for cancer.

Nanowire-induced electrostatic collection of urine EVs followed by in situ extraction of EV-encapsulated miRNAs.

(A) Schematic illustrations for urine EV collection and in situ extraction of urine EV–encapsulated miRNAs using a nanowire-anchored microfluidic device. (B) A schematic illustration (gray rods, nanowires; transparent cyan areas, PDMS) and an inset illustration on the lower left showing a cross-sectional image (yellow and blue represent nanowires and PDMS, respectively) for buried nanowires after poring, curing, and peeling off PDMS, and a vertical cross-sectional FESEM image of buried nanowires; nanowires and PDMS are highlighted as yellow and blue, respectively, and the white dotted line indicates a PDMS edge. Scale bar, 1 μm. (C) A schematic illustration and an inset illustration on the lower left showing a cross-sectional image for growing nanowires from the buried nanowires (nanowire-embedded PDMS), and a vertical cross-sectional image of the nanowire-embedded PDMS. Scale bar, 1 μm. (D) A schematic illustration and an inset illustration on the lower left showing a cross-sectional image for bonding the nanowire-embedded PDMS substrate to the microfluidic herringbone-structured PDMS substrate, an image of the nanowire-anchored microfluidic device (bonding the nanowire-embedded PDMS and the microfluidic herringbone-structured PDMS substrates) with PEEK tubes for an inlet and an outlet (scale bar, 1 cm), and a laser micrograph of the microfluidic herringbone structure on PDMS (scale bar, 1 mm). (E) A schematic illustration of the nanowire-embedded PDMS (gray rods, nanowires; transparent cyan areas, PDMS), and an overview of FESEM image for the nanowire-embedded PDMS (scale bar, 1 μm) after being exposed to lysis buffer. (F) A schematic illustration of nanowires on the Si substrate (gray rods, nanowires; dark cyan areas, Si substrate; faded cyan areas, Cr layer), and an overview of FESEM image for the nanowires on the Si substrate after being exposed to lysis buffer. Scale bar, 1 μm.

In situ extraction of miRNAs using the nanowire-anchored microfluidic device.

(A) Scatterplot of normalized intensities of miRNAs extracted from the collected EVs on nanowires in the device versus the ultracentrifuged EVs. Each point corresponds to a different miRNA type (that is, species). The boundary between pink and cyan represents the same level of miRNA expression for the two approaches. (B) Histogram of miRNA species for nanowires (pink) and ultracentrifugation extraction (cyan). Error bars show the SD for a series of measurements (n = 3). (C) Scatterplot of normalized intensities of miRNAs extracted from the collected EVs using the nanowire-anchored microfluidic device versus miRNA expression extracted from the collected EVs when using a commercially available kit. Each point corresponds to a different miRNA type (species). The boundary between pink and gray represents the same level of miRNA expression for the two approaches. (D) Histogram of miRNA species for nanowires (pink) and the commercially available kit (gray). Error bars show the SD for a series of measurements (n = 3). a.u., arbitrary units.

(A) A schematic illustration for the experimental process and calculation of collection efficiency. (B) Size distribution of the urinary free-floating objects in the untreated urine. Error bars show the SD for a series of measurements (n = 3). (C) Size distribution of the urinary free-floating objects in the flow-through fraction of the urine being processed by the device (pink) and in the ultracentrifuged urine (cyan). Error bars show the SD for a series of measurements (n = 3). (D) Fluorescently (PKH26) labeled EVs collected on nanowires. Red denotes PKH26-labeled EVs on nanowires. Scale bar, 500 μm. (E) An FESEM image of nanowires after introduction of PKH26-labeled EVs. White arrows indicate collected EVs. Scale bar, 200 nm. (F) Detection of EVs in urine on nanowires (pink) and a 96-well plate (cyan) using an antibody of CD63 or CD81. The measured concentration of the urinary free-floating objects was 1.4 × 108 ml−1. N.D. indicates fluorescence intensity was not detected. The black dotted line shows the signal level at 3 SD above the background. Error bars showing the SD for a series of measurements of nanowires and a 96-well plate (n = 24 and 3, respectively).

For intuitive understanding of the expression level of each miRNA and easy comparison between each group, we used color gradations showing signal intensity variation. Black, logarithmic signal intensity of 5; blue, logarithmic signal intensity less than or equal to 2; and yellow, logarithmic signal intensity greater than or equal to 8. Each column in the heat maps represents the logarithmic signal intensities of each miRNA corresponding to the color gradation.

Down-regulated and overexpressed miRNAs extracted from Fig. 4 between noncancer donors and each cancer donor.

Extracted miRNAs were the second smallest logarithmic signal intensities in one group larger than the three pulsing second largest logarithmic signal intensities in the other group. The symbols − and + show noncancer and cancer donors, respectively. Pink lines highlight minimums in one group that were larger than the three pulsing maximums in the other group, giving highlighted miRNAs an edge over other miRNAs. Green and orange lines highlight cancer-specific down-regulated miRNAs and overexpressed miRNAs, respectively.